Engineering Monoterpene Production in Yeast Using a Synthetic Dominant Negative Geranyl Diphosphate Synthase
Engineering Monoterpene Production in Yeast Using a Synthetic Dominant Negative Geranyl Diphosphate Synthase
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DOI：
10.1021/sb400115e
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发表时间：
2014-05-01
影响因子：
4.7
通讯作者：
Kampranis, Sotirios C.
中科院分区：
文献类型：
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作者：
Ignea, Codruta;Pontini, Marianna;Kampranis, Sotirios C.
单萜在食品和化妆品工业中具有既定的用途，并且最近还发现了作为高级生物燃料的应用。虽然代谢工程的努力迄今已取得了显着的产量较大的萜烯，单萜的生产力是落后的。在此，我们着手在酿酒酵母中建立单萜特异性生产平台，并确定酵母法呢基二磷酸合酶Erg20 p的顺序反应机制是限制单萜产量的重要因素。为了克服这一障碍，我们将Erg20p工程化到香叶基二磷酸合酶中，并实现了单萜滴度的显著增加。为了进一步提高产量，我们将工程化的香叶基二磷酸合酶转化为显性负性形式，以降低内源性Erg20 p作为法呢基二磷酸合酶发挥作用的能力，而不完全消除甾醇生物合成。合成的显性负Erg20p变体与萜烯合酶的融合，结合酵母菌株工程，进一步提高了单萜产量，并实现了比起始菌株的桧烯产量总体增加340倍。本文所述的设计可以容易地并入任何专用酵母菌株，而开发的质粒载体和杂合ERG 20缺失酵母菌株也可以用作酶表征和单萜途径阐明的即插即用系统。
Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting rnonoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.